This invention relates to the field of gel electrophoresis, particularly to pre-cast polyacrylamide gels having an extended shelf-life.
Gel electrophoresis is an important analytical and preparative separation technique in which charged molecules are separated under the influence of an electric field with a gel being used as the support matrix. This technique is particularly suitable for the separation of biological macromolecules. The gels commonly used in this technique are composed of polyacrylamide or agarose. Polyacrylamide gels are used particularly for the separation of biomolecules such as proteins, peptides, DNA, RNA, lipids, charged carbohydrates and the like, either naturally occurring or synthetic, in which the acrylamide is used in slab form being pre-cast prior to use. Traditionally, polyacrylamide gels have been prepared individually prior to use by polymerising an acrylamide/cross-linker solution in a gel-casting cassette to form a slab. Following electrophoresis, the gels are removed from the cassette, and the biomolecules are stained and/or transferred from the gel to another medium so that the separated biomolecules may be visualised, identified, recovered or quantified. Conventional polyacrylamide gels have the disadvantage of being relatively unstable and have a limited shelf-life.
As gels are often prepared on an individual basis prior to use, there can be variations between gels that have been cast separately such that direct comparisons between separations using different gels are not reliable. Furthermore, the monomer components in polyacrylamide gels are relatively toxic and continued preparation of gels increases the potential of exposure of these toxic monomers to the operator. There has now been a move to the commercial preparation of pre-cast gels under controlled conditions providing consistent and stable characteristics between batches of gels. Unfortunately, most pre-cast commercial gels still have the problem of limited shelf-life and must be used within a relatively short period of time to ensure accurate and reliable separations.
Currently, a major limitation in the production and sale of pre-cast polyacrylamide electrophoresis gels is the relatively short shelf-life, usually up to about three months. This is thought to be due to the hydrolysis of the amide groups in polyacrylamide to the carboxylic acid derivative in alkaline conditions [Geisthardt and Kruppa, Polyacrylamide Gel Electrophoresis: Reaction of Acrylamide at Alkaline pH with Buffer Components and Proteins Anal. Biochem. 160, 184-191 (1987)]. This hydrolysis is manifested in the gels as a loss of resolution of separated molecules, change in the migration distances of the separated molecules and reduced intensity of protein staining.
Typically, gels are prepared using alkaline buffers and run under alkaline conditions, usually around pH 8.9. A buffer system using Tris(hydroxymethyl)aminomethane and hydrochloric acid (Tris-HCl) developed by Laemmli [Cleavage of Structural Proteins During the Assembly of the Head of Bacteriophage T4 Nature 227, 680-686 (1970)], is a typical choice for xe2x80x9cstandardxe2x80x99 polyacrylamide gels in denaturing conditions. Although loss of stability in polyacrylamide gels occurs faster in alkaline conditions, this was thought to be unavoidable in standard gels. In principle, if the pH of the buffer in which the polymer network is formed could be lowered to around neutral, then the hydrolysis of the gel should be greatly reduced and so the gels should remain stable and useful for a longer period of time. Unfortunately, it has been difficult to find inexpensive chemical systems with an effective buffering capacity around neutral pH that are compatible with a polyacrylamide medium and suitable for gel electrophoresis and which do not have any effects that would cause a loss of stability through other interactions.
The most common buffer system used for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is that published by Ormstein [Disc Electrophoresis, 1, Background and Theory Ann. New York Acad. Sci. 121, 321-349 (1964)] and modified by Laemmli. This system uses a discontinuous electrophoresis system composed of a xe2x80x98stackingxe2x80x99 gel with a Tris concentration of 0.125 M at pH 6.8 and a xe2x80x98resolvingxe2x80x99 gel with a Tris concentration of 0.375 M at pH 8.8. The change in pH causes the proteins in the gel to xe2x80x98stackxe2x80x99 or concentrate into a fine line in the lower pH gel and then xe2x80x98resolvexe2x80x99 or spread out in the higher pH gel. The stacking gel is a short zone and the resolving gel is a longer zone in the gel. The other common Tris-HCl system is that of Schagger and von Jagow [Tricine-Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis for the Separation of Proteins in the Range from 1 to 100 kDa Anal. Biochem 166, 368-379 (1987)] who used a stacking gel of 0.75 M Tris at pH 8.45 and a resolving gel of 0.9 M at pH 8.45. Some reports have been made of the use of the Tris-HCl system below these pH values. Reisfield and Williams [Disc Electrophoresis in Polyacrylamide Gels: Extension to New Conditions of pH and Buffer Ann. N.Y. Acad. Sci. 121, 373-381 (1964)] have used Tris-HCl at a pH of 7.5, but their Tris concentration was very low (around 0.08 M) and their electrode buffer was Tris-diethylbarbituric acid. The present inventors have found that using Tris at these very low concentrations and at low pHs in more common electrophoresis of buffers leads to a distortion of bands and lack of resolution. King et al [Electrophoretic Conditions for High Resolution Citrus Isozymes in Polyacrylamide Gel Electrophoresis Electrophoresis 16, 32-38 (1995)] have used traditional concentrations of Tris (0.375 M) at pH 7.5. To reach the lower pH levels at this high concentration of Tris involves the addition of a large amount of hydrochloric acid leading to high conductivity in the gel. When the conductivity of the gel is high, the gels run very slowly in traditional electrode buffers.
A number of different gel buffer systems have been proposed for use at or around neutral pH that do not involve the use of Tris-HCl. The most common buffer system at neutral pH is the phosphate system of Shapiro [Biochem. Biophys. Res. Commun. 28, 815 (1967)] modified by Weber and Osborn [The Reliability of Molecular Weight Determinations by Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis J. Biol. Chem. 244(16), 4406-4412 (1969)]. Other buffer systems include Bis-Tris-HCl [Moos et al Reproducible High Yield Sequencing of Proteins Electrophoretically Separated and Transferred to an Inert Support J. Biol. Chem. 263(13), 6005-6008 (1988)], Tris-Acetate [Patton et al Tris-Tricine and Tris-Borate Buffer Systems Provide Better Estimates of Human Mesothelial Cell Intermediate Filament Protein Molecular Weights than the Standard Tris-Glycine System Anal. Biochem. 197, 25-33 (1991)], glycylglycine-NaOH [Hoffmann and Chalkley A Neutral pH Acrylamide Gel Electrophoretic System for Histones and Other Basic Proteins Anal. Biochem. 76, 539-546 (1976)], 1,4 piperazine-bis-(ethane sulfonic acid) (PIPES) xe2x80x94Na3PO4 [Davis and Gregerman Separation of Thyroxin (T4)-Binding Proteins of Human Serum in Polyacrylamide Gel at pH 7.4. I. Effect of pH on Distribution of Tracer Quantities of T4 J. Endocr. 30, 237-245 (1970)], 4-(2-hydroxyethyl)piperazine-1-ethanesulphonic acid (HEPES)/2-(N-morpholino)ethanesulphonic acid (MES)/3-(N-morpholino)propane sulphonic acid (MOPS)/Bicine-NaOH [12], MOPS-KOH [Thomas and Hodes A New Discontinuous Buffer System for the Electrophoresis of Cationic Proteins at Near-Neutral pH Anal. Biochem. 118, 194-196 (1981)] and Histidine-HCl/Tris-Citrate [King et al Electrophoretic Conditions for High Resolution Citrus Isozymes in Polyacrylamide Gel Electrophoresis Electrophoresis 16, 32-38 (1995)].
The buffering capacity of Tris-HCl is reduced at neutral pH and so commercially available pre-cast gels with longer shelf lives employ different buffer systems. U.S. Pat. No. 3,948,743 (Bio-Rad Laboratories, 1976) discloses the use of a strongly ionisable neutral salt in a concentration of 0.0005 N to 1.0 N at a pH of between 6 and 8. The neutral salt is preferably ammonium sulfate. The gels are then xe2x80x98pre-runxe2x80x99 in the buffer that is desired for separation to remove the salt before application of the sample. It would appear, however, that this method has not been commercialised due to the inconvenience of having to pre-run the gels before they are useful for electrophoresis.
U.S. Pat. No. 4,415,655 and U.S. Pat. No. 4,481,094 (TechAmerica Group Inc., 1983 and 1984) disclose the use of a salt of 2-amino-2-methyl-1,3-propanediol at a pH of 6.4 to 7.3 in combination with 2-amino-2-methyl-1,3-propanediol turine as an electrolyte buffer at a pH of 8.0 to 10.0.
U.S. Pat. No. 5,578,180 and U.S. Pat. No. 5,922,185 (Novel Experimental Technology, 1996 and 1999) disclose gels containing a buffer comprised of a primary organic amine or substituted amine with a pKa near neutrality, titrated with hydrochloric acid or acetic acid to a pH between 5.5 and 7.5. The amine is preferably Bis-(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris). The system runs with the gel and buffer at a pH around neutral. U.S. Pat. No. 6,059,948 is a continuation-in-part of these patents and discloses the use of a similar gel buffer consisting of an amine with pKa near neutrality and a zwitterionic base with a pKb between 6 and 9 with the buffer having a pH between pH 5.5 and 7.5.
U.S. Pat. No. 5,464,516 (Hymo Corporation, Atto Corporation, 1995) discloses gels which contain a buffer comprised of an acid, amine and an ampholyte that has the same number of anionic and cationic groups in each single molecule. The pH of these gels is between 4 and 7.5 and have been designed to give a wide separation range and stability. This patent mentions that lowering the pH and Tris concentration in conventional gel systems does not allow sufficient movement of proteins so is not suitable for use in electrophoretic separation of compounds.
The present inventors have now surprisingly found that by manipulating the conventional Tris-HCl buffer system, stable gels can be prepared that have comparable separation characteristics as standard gels but having the advantage of long shelf-life.
The present invention relates a gel system with a substantially neutral gel buffer system which assists in limiting the hydrolysis of the polymer network of the gel. Modification of conventional Tris-HCl buffer systems resulted in increased stability of the gel during storage. Importantly, in use the gels have minimal variation in migration distances of the proteins and increased retention of band sharpness over time.
In a first aspect, the present invention provides a polyacrylamide gel utilising a buffer system comprising Tris(hydroxymethyl)aminomethane at a concentration range of about 0.15 to 0.25 M titrated with hydrochloric acid to a pH between about 6.5 and 7.5.
Preferably, the gel comprises Tris(hydroxymethyl)aminomethane at about 0.18 to 0.22 M and having a pH of about 6.8 to 7.2. More preferably, the gel comprises Tris(hydroxymethyl)aminomethane at about 0.20 M and having a pH of about 7.0.
The gels produced according to the present invention are substantially relatively stable to polyacrylamide hydrolysis during storage for at extended periods. The gels have an acceptable shelf-life of at least about six months, preferably at least about nine months and even more preferably at least about 12 months after storage at about or around 4xc2x0 C. An acceptable shelf-life can be determined by the gel producing a resolving protein separation migration pattern under electrophoresis conditions which is similar to or better than the degree of resolution of the same or similar sample using standard Tris/HCl gels. Typically, there is little or no significant deterioration in the degree of separation or sharpness of protein bands in the gels after prolonged storage times prior to use.
The polyacrylamide gels according to the present invention are cross-linked acrylamide formed by treating acrylamide with a cross-linking agent, usually N,Nxe2x80x2-methylene-bis-acrylamide (Bis)under suitable initiating conditions. Although Bis is the cross-linker of choice for most standard gel-forming processes, a number of other cross-linking agents have been developed or are being developed. Examples include Piperazine diacrylamide (PDA) and N,Nxe2x80x2diallyl-tartardiamide (DATD). The present invention is therefore applicable to all forms of polyacrylamide, irrespective of the cross-linking agent.
In a second aspect, the present invention provides a method of preparing a polyacrylamide gel, the method comprising polymerising acrylamide in the presence of a cross-linking agent, water, a buffer system for the polyacrylamide gel and a polymerisation means;
wherein the buffer system comprises Tris(hydroxymethyl)aminomethane at the concentration range of about 0.15 to 0.25 M titrated with hydrochloric acid to a pH between about 6.5 and 7.5.
Preferably, the gel comprises Tris(hydroxymethyl)aminomethane at about 0.18 to 0.22 M and having a pH of about 6.8 to 7.2. More preferably, the gel comprises Tris(hydroxymethyl)aminomethane at about 0.20 M and having a pH of about 7.0.
The gels produced according to the present invention are substantially relatively stable to polyacrylamide hydrolysis during storage for at extended periods. The gels have an acceptable shelf-life of at least about six months, preferably at least about nine months and even more preferably at least about 12 months after storage at about or around 4xc2x0 C. An acceptable shelf-life can be determined by the gel producing a resolving protein separation migration pattern under electrophoresis conditions which is similar to or better than the degree of resolution of the same or similar sample using standard Tris/HCl gels. Typically, there is little or no significant deterioration in the degree of separation or sharpness of protein bands in the gels after prolonged storage times prior to use.
The polyacrylamide gels according to the present invention are cross-linked acrylamide formed by treating acrylamide with a cross-linking agent, usually N,Nxe2x80x2-methylene-bis-acrylamide (Bis)under suitable initiating conditions. Although Bis is the cross-linker of choice for most standard gel-forming processes, a number of other cross-linking agents have been developed or are being developed. Examples include Piperazine diacrylamide (PDA) and N,Nxe2x80x2diallyl-tartardiamide (DATD). The present invention is therefore applicable to all forms of polyacrylamide, irrespective of the cross-linking agent.
Preferably, the polymerisation means is by redox type initiator using ammonium persulphate (APS) and N,N,Nxe2x80x2,Nxe2x80x2-tetramethylethelenediamine (TEMED). Other free-radical initiator systems suitable for polymerising acrylamide gels including redox, thermal, photoactivation systems would also be suitable for the present invention.
In a third aspect, the present invention provides an apparatus for use in gel electrophoresis, the apparatus comprising a gel according to the first aspect of the present invention adapted to be inserted in an electrophoresis apparatus.
The apparatus can be any standard electrophoresis apparatus housing a gel according to the present invention. The apparatus typically comprises one or more receptacles for electrophoresis buffer electrodes positioned in contact with the electrode buffer, and a housing to accept an arrangement for the electrophoresis gel, typically a cassette arrangement.
In a fourth aspect, the present invention provides a method of performing electrophoresis, the method comprising:
(a) applying a sample containing one or more compounds to be separated to the gel of an electrophoresis apparatus according to the third aspect of the present invention;
(b) providing an electrode buffer; and
(b) subjecting the gel to an electric field for sufficient time such that at least one compound in the sample is caused to move into the gel.
In one preferred form, the electrode buffer comprises Tris(hydroxymethyl) aminomethane and 4-(2-hydroxyethyl)piperazine-1-ethanesulphonic acid (HEPES). Preferably, the electrode buffer has a concentration of about 0.05 to 0.125 M and has a pH of about 7.5 to 8.5.
Throughout this specification, unless the context requires otherwise, the word xe2x80x9ccomprisexe2x80x9d, or variations such as xe2x80x9ccomprisesxe2x80x9d or xe2x80x9ccomprisingxe2x80x9d, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application.
In order that the present invention may be more clearly understood preferred forms will be described with reference to the following examples and drawings.